Regulation of gene expression in keratinocytes, and epithelial cells in general, is poorly understood. Understanding the regulatory mechanisms involved in this process is critical to understanding the coordinated expression of numerous unlinked genes that is the basis of embryonic development and differentiation which produces the epidermal phenotype. in order to understand the molecular mechanisms involved in these two fundamental processes, one must focus on the study of specific prototypic genes. We have recently identified a novel keratinocyte protein, epidermal surface antigen or ESA, that seems well suited for this purpose, since: normally differentiating keratinocytes express high levels of ESA protein; ESA expression occurs very early in embryonic development; ESA protein expression is not limited to one epidermal layer but extends throughout all suprabasal layers of the epidermis; the ESA gene is well conserved in mammalian species; and ESA expression may be essential for epidermal adherence. The goal of the studies described in this proposal is to provide a complete understanding of the transcriptional regulation of the ESA gene in keratinocytes. To guide the design and conduct of experiments to achieve this goal, we PROPOSE THE HYPOTHESIS THAT ESA GENE EXPRESSION IS CONTROLLED BY THE INTERPLAY OF REGULATORY REGIONS OF THE GENE WITH TISSUE-SPECIFIC NUCLEAR PROTEINS. To prove or disprove this hypothesis, we propose the following specific aims. We will (1) isolate the complete ESA gene; (2) analyze its structural and regulatory regions; (3) identify the cis-acting elements that regulate its tissue specific expression in keratinocytes; and (4) identify the keratinocyte nuclear proteins involved in regulation of ESA expression. In addition, recent studies have shown that ESA expression is not limited-to the epidermis but also occurs in simple epithelia, melanocytes, and in some squamous cell carcinoma and melanoma cell lines. Therefore, once we have completed specific aims (1)-(4) in keratinocytes, we can study ESA expression in other cells to verify which regulatory mechanisms are tissue specific and which occur in all cell types that express the ESA gene. These findings will be important for understanding the development and maintenance of a differentiated epidermis, and ultimately the pathogenesis of epidermal disease.